Protein phosphorylation by intracellular kinases is one of the most critical reactions in signaling within cells and is known to play important roles in various processes such as survival, proliferation, and differentiation of cells (Cell 100, 113-127 (2000)). Protein kinases catalyze transfer of the γ-phosphate of ATP and phosphorylation of hydroxyl groups of serines, threonines and/or tyrosines on the substrate proteins, and upon such phosphorylation, substrate proteins are subject to conformational changes due to negative charges of the phosphates, which subsequently triggers their enzymatic activation and interaction with their respective target proteins. Therefore, it is expected that by screening substances that enhance or suppress intracellular signaling triggered by protein phosphorylation and dephosphorylation, not only may diagnosis of diseases become possible, but important information for the development of new drugs may be obtained, as well.
Conventionally, analysis of signaling related to the kinase proteins has been preformed using means such as electrophoresis, immunocytochemistry, and kinase assay in vitro. However, these conventional methods are destructive methods and could not provide information on spatial and temporal analysis of signals from protein phosphorylation and dephosphorylation in living cells.
In contrast, unlike kinase signaling, second messenger signaling such as Ca2+ (Nature 388, 882-887 (1997), inositol 1,4,5-triphosphate (Science 248, 1527-1530 (1999)), diacylglycerol (J. Cell Biol. 140, 485-498 (1998)), cyclic AMP (Nature 349, 694-697 (1997); Nat. Cell Biol. 2, 25-29 (1999)) and cyclic GMP (Anal. Chem. 72, 5918-5924 (2000)) has been visualized using fluorescent indicators; it has been reported that in such measurement methods, highly accurate spatial and temporal analysis of second messenger signaling in single living cells is made possible (Curr. Opinion Neurobiol. 10, 416-421 (2000)).
In recent years, along with probes for visualizing second messenger signaling, probes for visualizing kinase signaling in living cells have been studied and a few have been reported (Anal. Biochem. 195, 148-152 (1991); NeuroReport 7, 2695-2700 (1996); FEBS Lett. 414, 55-60 (1997); Nat. Biotechnol. 18, 313-316 (2000)). However, these imaging probes are all based on conformational changes of the substrate peptides themselves upon phosphorylation. Because controlling such conformational changes is impossible, such probes were only applicable to specific kinase signaling and lacked practicality.
Accordingly, the invention of the present patent application has been made in view of the above problems, and the object of the present invention is to provide a practical method for the detection and measurement of protein phosphorylation and dephosphorylation in living cells, animal bodies, plant bodies etc., that enables a non-destructive method for monitoring and further enables spatial and temporal analysis, thereby solving the problems of conventional techniques.